Gene therapy involves the introduction of genetic material into host cells with the goal of treating or curing disease. Many diseases are caused by “defective” genes that result in a deficiency in an essential protein. One approach for correcting faulty gene expression is to insert a normal gene (transgene) into a nonspecific location within the genome to replace a nonfunctional, or “defective,” disease-causing gene. Gene therapy can also be used as a platform for the delivery of a therapeutic protein or RNA to treat various diseases so that the therapeutic product is expressed for a prolonged period of time, eliminating the need for repeat dosing. A carrier molecule called a vector must be used to deliver a transgene to the patient's target cells, the most common vector being a virus that has been genetically altered to carry normal human genes. Viruses have evolved a way of encapsulating and delivering their genes to human cells in a pathogenic manner and thus, virus genomes can be manipulated to insert therapeutic genes.
Stable transgene expression can be achieved following in vivo delivery of vectors based on adenoviruses or adeno-associated viruses (AAVs) into non dividing cells, and also by transplantation of stem cells transduced ex vivo with integrating and non-integrating vectors, such as those based on retroviruses and lentiviruses. AAV vectors are used for gene therapy because, among other reasons, AAV is nonpathogenic, it does not elicit a deleterious immune response, and AAV transgene expression frequently persists for years or the lifetime of the animal model (see Shyam et al., Clin. Microbiol. Rev. 24(4):583-593). AAV is a small, nonenveloped human parvovirus that packages a linear strand of single stranded DNA genome that is 4.7 kb. Productive infection by AAV occurs only in the presence of a helper virus, either adenovirus or herpes virus. In the absence of a helper virus, AAV integrates into a specific point of the host genome (19q 13-qter) at a high frequency, making AAV the only mammalian DNA virus known to be capable of site-specific integration. See, Kotin et at., 1990, PNAS, 87: 2211-2215. However, recombinant AAV, which does not contain any viral genes and only a therapeutic gene, does not integrate into the genome. Instead the recombinant viral genome fuses at its ends via inverted terminal repeats to form circular, episomal forms which are predicted to be the primary cause of the long term gene expression (see Shyam et at., Clin. Microbiol. Rev. 24(4):583-593).
Virtually all pre-clinical and clinical applications of gene therapy have used vectors that express the transgene from a constitutive promoter, which means it is active at a fixed level for as long as the vector genome persists. However, many diseases that are amenable to gene therapy may need to have expression of the transgene regulated. Several systems have been described which that are based on the general principle of placing a gene of interest under the control of a drug-inducible engineered transcription factor in order to positively induce gene expression (Clackson et at., 1997, Curr Opin Chem Biol, 1 (2): 210-8; Rossi et at., Curr Opin Biotechnol, 1998. 9(5): p. 451-6). The various systems can be divided into two classes. In the first, a DNA-binding domain that is allosterically regulated by inducers such as tetracyclines, antiprogestins, or ecdysteroids is coupled to a transactivation domain. The addition (or in some cases removal) of the drug leads to DNA binding and hence transcriptional activation. In the second, allosteric control is replaced with the more general mechanism of induced proximity. DNA binding and activation domains are expressed as separate polypeptides that are reconstituted into an active transcription factor by addition of a bivalent small molecule, referred to as a chemical inducer of dimerization or “dimerizer.” While these systems are useful in gene therapy systems that require inducing transgene expression, they have not addressed the need to be able to turn off or permanently ablate transgene expression if it is no longer needed or if toxicity due to long-term drug administration ensues.